# US 10,308,955 B2 · Transient expression vectors, preparation and uses thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,308,955 B2
- Document ID: US10308955B2
- Status: Granted
- Kind: B2
- Application number: 14/352,902
- Filed: 2012-10-26
- Published: 2014-09-25
- Granted: 2019-06-04
- Earliest claimed: 2011-10-26
- Assignees: Newvectys
- Applicants: Newvectys
- Inventors: Chamsy Sarkis; Dorothee Altemir; Nicolas Grandchamp; Stephanie Philippe
- Web page: https://patentgrep.com/patent/US10308955B2

## CPC

- C12N 15/86: Viral vectors
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention describes a recombinant retroviral vector which cannot by itself achieve complete reverse transcription as well as its uses, in particular for transiently transferring in vitro, ex vivo or in vivo at least one ribonucleic acid sequence of interest in a cell. Such a transient transgene expression is of interest in the context of research, therapy and more generally in the field of biotechnology.

## Claims

### Claim 1

A recombinant HIV-1 vector which cannot on its own achieve complete reverse transcription wherein the vector is a recombinant ribonucleic vector genome comprising (i) an intact 5′ LTR retroviral sequence and a 3′ LTR retroviral sequence flanking: an intact primer binding site (PBS) sequence, a retroviral psi encapsidation sequence and at least one transgene, and (ii) a coding sequence for a reverse transcriptase which is non-functional for completing reverse transcription, wherein the non-functional reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at position 110 of SEQ ID NO: 1 and/or a mutation affecting the RNAseH domain at position 478 of SEQ ID NO: 1, and wherein formation of DNA from the vector is prevented due to the presence of the non-functional reverse transcriptase.

### Claim 2 (depends on 1)

The vector according to claim 1, wherein the reverse transcriptase comprises a mutation affecting polymerase activity and a mutation affecting RNAseH activity.

### Claim 3 (depends on 1)

The vector according to claim 1, wherein the reverse transcriptase contains a mutation at position 110 of SEQ ID NO: 1 and/or a mutation at position 478 of SEQ ID NO: 1, the mutation being a substitution of the amino acid D at position 110 of SEQ ID NO: 1 by a naturally occurring amino acid residue selected from A, C, E, F, G, H, 1, K, L, M, N, P, Q, R, S, T, V, W, and Y, and/or a substitution of the amino acid E at position 478 of SEQ ID NO: 1 by a naturally occurring amino acid residue selected from A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

### Claim 4 (depends on 3)

The vector according to claim 3, wherein the reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at positions 110 of SEQ ID NO: 1 and a mutation affecting the RNAseH domain at position 478 of SEQ ID NO: 1.

### Claim 5 (depends on 1)

The vector according to claim 1, wherein the recombinant vector genome further comprises at least one post-transcriptional regulatory sequence selected from WPRE, APP 5′UTR, TAU 3′UTR, and a miRNA target sequence.

### Claim 6 (depends on 1)

The vector according to claim 1, wherein the transgene is selected from a catalytic nucleic acid, an aptamer, a miRNA, a decoy RNA, or a nucleic acid encoding a biologically active peptide.

### Claim 7 (depends on 6)

The vector according to claim 6, wherein said biologically active peptide is an enzyme, a transcription factor, a growth factor, a trophic factor, a hormone, a cytokine, an antibody, a receptor, a differentiation factor, a colony stimulation factor, a suicide protein, a cell-cycle modifying protein, an anti-proliferative protein, a nuclease, a recombinase, transposase, a neurotransmitter or a precursor thereof.

### Claim 8

A method for producing an HIV-1 vector comprising expressing within a cell the following transcomplementation elements:
i) a transcomplementation capsid cassette, optionally split into several cassettes, comprising sequences derived from an HIV-1 genome encoding a retroviral gag sequence, said transcomplementation capsid cassette lacking any functional psi encapsidation signal, wherein said transcomplementation capsid cassette comprises a retroviral pol sequence encoding a reverse transcriptase which is non-functional for complete reverse transcription wherein the non-functional reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at position 110 of SEQ ID NO: 1 and/or a mutation affecting the RNAseH domain at position 478 of SEQ ID NO: 1, and wherein said transcomplementation capsid cassette comprises an intact primer binding site (PBS) sequence,
ii) a transcomplementation envelope cassette encoding for an envelope glycoprotein, and
iii) a transcomplementation vector cassette encoding for an HIV-1 recombinant ribonucleic vector genome comprising an intact 5′ LTR retroviral sequence and a 3′ LTR retroviral sequence flanking a retroviral psi encapsidation sequence, at least one transgene and, optionally, at least one post-transcriptional regulatory sequence, and
recovering the HIV-1 vectors produced from the expression of the elements encoded by each cassette.

### Claim 9 (depends on 1)

A composition comprising a vector according to claim 1.

### Claim 10 (depends on 9)

The composition according to claim 9, wherein the composition is a pharmaceutical composition.

### Claim 11 (depends on 3)

The vector according to claim 3, wherein the mutation is at position 110.

### Claim 12 (depends on 3)

The vector according to claim 3, wherein the mutation is at position 478.

### Claim 13 (depends on 4)

The vector according to claim 4, wherein the mutations are a D110E and a E478Q substitution.

### Claim 14 (depends on 8)

The method according to claim 8, wherein the non-functional reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at position 110 of SEQ ID NO: 1 and/or a mutation affecting the RNAseH domain at position 478 of SEQ ID NO: 1.